Cleft palate is among the most common birth defects in humans. We and others have shown that transforming growth factor-3 (Tgf-3) plays a critical role in palatal epithelial fusion both in mice and in humans. Failure in Tgf-3 signaling leads to a condition in which the palatal medial edge epithelium fails to disappear, and subsequently fully- grown palatal shelves fail to fuse. During previous funding years of this grant, we have shown that Tgf- signaling is required both in the palatal epithelium and in the palatal mesenchyme for appropriate palatal shelf growth, patterning and fusion. Consequently, our results suggest that the role of Tgf-3 during palatogenesis is broader and more complex than previously thought. Based on the recent findings and our preliminary data, we have formulated an overall hypothesis that specific, modular, long-distance regulatory elements are responsible of targeting Tgf3 expression to the prefusion palatal epithelium. This epithelial-specific expression of Tgf3, in turn, induces both Smad4-dependent (canonical) and Smad4-independent (non-canonical) signaling events in the palatal epithelium that are together with the mesenchymal Tgf-3 signaling, required for successful palatal fusion. In specific aim 1, we propose to test the hypothesis that Tgf3 transcription in palatal medial edge epithelial cells is regulated by distant 5' and/or 3' enhancers. In specific aim 2, we propose to test the hypothesis that Tgf--activated kinase-1 (Tak1) functions together with the Smad4-mediated signaling pathway to induce Tgf-3-induced cell cycle arrest and subsequent apoptosis in palatal epithelial cells. In specific aim 3, we propose to test the hypothesis that in Tgfb3 null mutants, imbalance between Tgf- and Notch signaling leads to an attenuated expression of cell cycle inhibitors ultimately resulting in cleft palate, and finally, in specific aim 4, we propose to test the hypothesis that Tgf-3 plays a previously unexpected role in the palatal mesenchyme by regulating appropriate patterning of prefusion palatal shelves. Our unique experimental models and state-of-art strategy will allow us to define to the role of canonical and non-canonical Tgf- signaling during palatogenesis. Collectively, the proposed experiments are likely to be of critical importance in attempting to understand the molecular basis of the cleft palate syndrome in humans.